Fusion protein construct

Abstract

The disclosure provides constructs comprising a first fusion protein, a second fusion protein, and a linker, wherein the first fusion protein and the second fusion protein each include an affinity reagent and a reactive enzyme, and the linker includes a first and second functional groups specific for irreversibly inhibiting the first and second fusion protein reactive enzymes. The disclosure further provides a method including (a) contacting a first fusion protein including an affinity reagent and a reactive enzyme with a linker including a functional group specific for irreversibly inhibiting the first fusion protein reactive enzyme thereby coupling the first fusion protein and the linker, and (b) contacting a second fusion protein including an affinity reagent and a reactive enzyme with the linker, the linker including a functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme thereby coupling the second fusion protein and the linker.

What is claimed: 
     
       1. A construct comprising
 a first fusion protein, a second fusion protein, and a linker, 
 wherein
 the first fusion protein and the second fusion protein each comprises an affinity reagent and a reactive enzyme; and 
 the linker comprises a first functional group specific for irreversibly inhibiting the first fusion protein reactive enzyme at a first terminus, and a second functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme at a second terminus, wherein the first functional group specific for irreversibly inhibiting the first fusion protein reactive enzyme is coupled to the first fusion protein reactive enzyme and the second functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme is coupled to the second fusion protein reactive enzyme, such that the first fusion protein and second fusion protein are linked in the form of the construct, and 
 wherein the first fusion protein reactive enzyme comprises cutinase and the second fusion protein reactive enzyme comprises HaloTag or SnapTag; and 
 wherein the first fusion protein affinity reagent and the second fusion protein affinity reagent each comprise an antibody or fragment thereof, 
 wherein the antibody fragment comprises Fab′ fragments, F(ab)2 fragments, Fv fragments, Fc fragments, one or more complementarity determining regions (CDR) fragments, an individual heavy chain, an individual light chain, or a dimeric heavy and light chain. 
 
 
     
     
       2. The construct of  claim 1 , wherein the antibody or fragment thereof is selected from the group consisting of a light chain variable domain (V L ), a light chain constant domain (C L ), a heavy chain variable domain (V H ), a heavy chain constant domain (C H 1), and a combination thereof. 
     
     
       3. The construct of  claim 1 , wherein the antibody or fragment thereof is selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab, capromab, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab tiuxetan, muromonab, edrecolomab gemtuzumab ozogamicin, golimumab, certolizumab, eculizumab, ustekinumab, panitumumab, tositumomab, bevacizumab, raxibacumab, tocilizumab, brentuximab, ofatumumab, belimumab, ramucirumab, vedolizumab, obinutuzumab, pembrolizumab, ranibizumab, pertuzumab, denosumab, catumaxomab, golimumab, siltuximab, natalizumab, panitumumab, and denosumab. 
     
     
       4. The construct of  claim 1 , wherein the antibody or fragment thereof is a synthetic antibody domain. 
     
     
       5. The construct of  claim 1 , wherein the linker is a polyoxazoline, polyacrylomorpholine, polyvinylpyrrolidone, polyphosphazene, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, poly(1-hydroxymethylethylene hydroxymethyl formal) (“PHF”), a polyhydroxyalkylacrylate, 2-methyacryloyloxy-2′-ethyltrimethylammonium phosphate (“MPC”), or a structure selected from: 
       
         
           
           
               
               
           
         
         wherein: 
         m is 0-10; 
         n is 1-100; 
         each p independently is 0, 1, 2, 3, or 4; 
         q is 0, 1, or 2; 
         r is 1 or 2; 
         E is NH or CHR 10 ; 
         G is O, CH 2 , CHOH, CHNH 2 , CHCOOH, or CHSO 3 H; 
         R 10  is OH, NH 2 , or COOH; 
         each R 11  independently is H, OH, NH 2 , or COOH. 
       
     
     
       6. A method comprising
 (a) contacting a first fusion protein comprising an affinity reagent and a reactive enzyme comprising cutinase with a linker comprising a functional group specific for irreversibly inhibiting the first fusion protein reactive enzyme at a first terminus thereby coupling the first fusion protein reactive enzyme and the linker at the first terminus, and 
 (b) contacting a second fusion protein comprising an affinity reagent and a reactive enzyme comprising HaloTag or SnapTag with a second terminus of the linker, the second terminus of the linker comprising a functional group specific for irreversibly inhibiting the second fusion protein reactive enzyme thereby coupling the second fusion protein reactive enzyme and the linker at the second terminus, 
 thereby forming a construct comprising the first fusion protein coupled to the linker through the reactive enzyme of the first fusion protein and the functional group specific for irreversibly inhibiting the reactive enzyme of the first fusion protein and the second fusion protein coupled to the linker through the reactive enzyme of the second fusion protein and the functional group specific for irreversibly inhibiting the reactive enzyme of the second fusion protein; 
 wherein the first fusion protein affinity reagent and the second fusion protein affinity reagent each comprise an antibody or fragment thereof, 
 wherein the antibody fragment comprises Fab′ fragments, F(ab)2 fragments, Fv fragments, Fc fragments, one or more complementarity determining regions (CDR) fragments, an individual heavy chain, an individual light chain, or a dimeric heavy and light chain. 
 
     
     
       7. The method of  claim 6 , wherein steps (a) and (b) are performed sequentially. 
     
     
       8. The method of  claim 6 , wherein steps (a) and (b) are preformed contemporaneously. 
     
     
       9. The method of  claim 6 , wherein the linker is a of polyoxazoline, polyacrylomorpholine, polyvinylpyrrolidone, polyphosphazene, polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acid anhydride, poly(1-hydroxymethylethylene hydroxymethyl formal) (“PHF”), a polyhydroxyalkylacrylate, 2-methyacryloyloxy-2′-ethyltrimethylammonium phosphate (“MPC”), or a structure selected from: 
       
         
           
           
               
               
           
         
         wherein: 
         m is 0-10; 
         n is 1-100; 
         each p independently is 0, 1, 2, 3, or 4; 
         q is 0, 1, or 2; 
         r is 1 or 2; 
         E is NH or CHR 10 ; 
         G is O, CH 2 , CHOH, CHNH 2 , CHCOOH, or CHSO 3 H; 
         R 10  is OH, NH 2 , or COOH; 
         each R 11  independently is H, OH, NH 2 , or COOH. 
       
     
     
       10. A construct prepared by the method of  claim 6 . 
     
     
       11. A method comprising administering the construct of  claim 1  to a patient in need thereof. 
     
     
       12. The method of  claim 11 , wherein the patient suffers from breast cancer, inhalational anthrax, rheumatoid arthritis, systemic juvenile idiopathic arthritis, Hodgkin lymphoma, systemic anaplastic large cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B cell lymphoma, multiple sclerosis, systemic lupus erythematosus, gastric or gastro-esophageal junction adenocarcinoma, metastatic non-small-cell lung carcinoma, ulcerative colitis, Crohn's disease, follicular lymphoma, melanoma, macular degeneration, osteoporosis, treatment-induced bone loss, metastases to bone, giant cell tumor of bone, malignant ascites, psoriatic arthritis, ankylosing spondylitis, metastatic renal cell cancer, prostate cancer, ovarian cancer, colorectal cancer, multiple myeloma, and Castleman's disease. 
     
     
       13. The construct of  claim 1 , wherein the antibody or fragment thereof is a chimeric antibody, a human antibody, or a humanized antibody. 
     
     
       14. The construct of  claim 1 , wherein the antibody or fragment thereof comprises trastuzumab or a fragment thereof. 
     
     
       15. The method of  claim 6  wherein the antibody or fragment thereof is selected from the group consisting of a light chain variable domain (V L ), a light chain constant domain (C L ), a heavy chain variable domain (V H ), a heavy chain constant domain (C H 1), and a combination thereof. 
     
     
       16. The method of  claim 6 , wherein the antibody or fragment thereof is a chimeric antibody, a human antibody, or a humanized antibody. 
     
     
       17. The method of  claim 6 , wherein the antibody or fragment thereof comprises trastuzumab or a fragment thereof. 
     
     
       18. The method of  claim 6 , wherein the antibody or fragment thereof is selected from the group consisting of adalimumab, alemtuzumab, arcitumomab, cetuximab, trastuzumab, imciromab, capromab, infliximab, abciximab, rituximab, basiliximab, palivizumab, nofetumomab, omalizumab, daclizumab, ibritumomab tiuxetan, muromonab, edrecolomab gemtuzumab ozogamicin, golimumab, certolizumab, eculizumab, ustekinumab, panitumumab, tositumomab, bevacizumab, raxibacumab, tocilizumab, brentuximab, ofatumumab, belimumab, ramucirumab, vedolizumab, obinutuzumab, pembrolizumab, ranibizumab, pertuzumab, denosumab, catumaxomab, golimumab, siltuximab, natalizumab, panitumumab, and denosumab. 
     
     
       19. The method of  claim 6 , wherein the antibody or fragment thereof is a synthetic antibody domain.